技术资料

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor,including ULBP-1,-2,and -3,but not MICA or MICB,in infected cells both in vitro and in vivo. However,the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G(2) cell-cycle arrest,conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4(+) T lymphocytes by a process that is Vpr dependent. Importantly,Vpr enhanced the susceptibility of HIV-1-infected cells to NK cell-mediated killing. Strikingly,Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell-mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells,suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall,these results indicate that Vpr is a key determinant responsible for HIV-1-induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced CD4(+) T-lymphocyte depletion but may also take part in HIV-1-induced NK-cell dysfunction. View Publication

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study,we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs,characterized by transcriptional repression of IL-1α and β,IL-1R1,and vascular endothelial growth factor A,and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs,therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated,at least in part,by increases in EPC/CAC proliferation,by decreases in EPC/CAC apoptosis,and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs,and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis,but not anti-neutrophil cytoplasmic Ab-positive vasculitis,showed this pathway to be operational in vivo,with increased IL-1R antagonist,downregulation of vascular endothelial growth factor A,and glomerular and blood vessel decreased capillary density,compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease. View Publication

Neutrophil apoptosis is essential for the resolution of inflammation but is delayed by several inflammatory mediators. In such terminally differentiated cells it has been uncertain whether these agents can inhibit apoptosis through transcriptional regulation of anti-death (Bcl-X(L),Mcl-1,Bcl2A1) or BH3-only (Bim,Bid,Puma) Bcl2-family proteins. We report that granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α prevent the normal time-dependent loss of Mcl-1 and Bcl2A1 in neutrophils,and we demonstrate that they cause an NF-κB-dependent increase in Bcl-X(L) transcription/translation. We show that GM-CSF and TNF-α increase and/or maintain mRNA levels for the pro-apoptotic BH3-only protein Bid and that GM-CSF has a similar NF-κB-dependent effect on Bim transcription and BimEL expression. The in-vivo relevance of these findings was indicated by demonstrating that GM-CSF is the dominant neutrophil survival factor in lung lavage from patients with ventilator-associated pneumonia,confirming an increase in lung neutrophil Bim mRNA. Finally GM-CSF caused mitochondrial location of Bim and a switch in phenotype to a cell that displays accelerated caspase-9-dependent apoptosis. This study demonstrates the capacity of neutrophil survival agents to induce a paradoxical increase in the pro-apoptotic proteins Bid and Bim and suggests that this may function to facilitate rapid apoptosis at the termination of the inflammatory cycle. View Publication

Rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles that code a five-amino acid sequence motif in positions 70-74 of the HLA-DRbeta-chain,called the shared epitope (SE). The mechanistic basis of SE-RA association is unknown. We recently found that the SE functions as an allele-specific signal-transducing ligand that activates an NO-mediated pathway in other cells. To better understand the role of the SE in the immune system,we examined its effect on T cell polarization in mice. In CD11c(+)CD8(+) dendritic cells (DCs),the SE inhibited the enzymatic activity of indoleamine 2,3 dioxygenase,a key enzyme in immune tolerance and T cell regulation,whereas in CD11c(+)CD8(-) DCs,the ligand activated robust production of IL-6. When SE-activated DCs were cocultured with CD4(+) T cells,the differentiation of Foxp3(+) T regulatory cells was suppressed,whereas Th17 cells were expanded. The polarizing effects could be seen with SE(+) synthetic peptides,but even more so when the SE was in its natural tridimensional conformation as part of HLA-DR tetrameric proteins. In vivo administration of the SE ligand resulted in a greater abundance of Th17 cells in the draining lymph nodes and increased IL-17 production by splenocytes. Thus,we conclude that the SE acts as a potent immune-stimulatory ligand that can polarize T cell differentiation toward Th17 cells,a T cell subset that was recently implicated in the pathogenesis of autoimmune diseases,including RA. View Publication

Coinfection of HIV-1 patients with Plasmodium falciparum,the etiological agent of malaria,results in a raise of viral load and an acceleration of disease progression. The primary objective of this study was to investigate whether the malarial pigment hemozoin (HZ),a heme by-product of hemoglobin digestion by malaria parasites,can affect HIV-1 transmission by monocytes-derived dendritic cells (DCs) to CD4(+) T cells when HZ is initially internalized in monocytes before their differentiation in DCs. We demonstrate in this study that HZ treatment during the differentiation process induces an intermediate maturation phenotype when compared with immature and fully mature DCs. Furthermore,the DC-mediated transfer of HIV-1 is enhanced in presence of HZ,a phenomenon that may be linked with the capacity of HZ-loaded cells to interact and activate CD4(+) T cells. Altogether our findings suggest a new mechanism that could partially explain the increased HIV-1 virus production during a coinfection with P. falciparum. Understanding the multifaceted interactions between P. falciparum and HIV-1 is an important challenge that could lead to the development of new treatment strategies. View Publication

Macrophages have a central role in the pathogenesis of cryptococcosis since they are an important line of defense,serve as a site for fungal replication,and also can contribute to tissue damage. The objective of this study was to investigate the interaction of macrophages with cells from smooth-colony variants (SM) and mucoid-colony variants (MC) arising from phenotypic switching of Cryptococcus neoformans. Alveolar macrophages (AMs) isolated from SM- and MC-infected mice exhibited differences in gene and surface expression of PD-L1,PD-L2,and major histocompatibility class II (MHC-II). PD-L1 and PD-L2 are the ligands for PD1 and are differentially regulated in Th1- and Th2-type cells. In addition,macrophage activation in SM- and MC-infected mice was characterized as alternatively activated. Flow cytometric and cytokine analysis demonstrated that MC infection was associated with the emergence of Th17 cells and higher levels of interleukin-17 (IL-17) in lung tissue,which were reduced by AM depletion. In conclusion,our results indicate that macrophages play a significant role in maintaining damage-promoting inflammation in the lung during MC infection,which ultimately results in death. View Publication

The NKG2D receptor activates natural killer (NK) cell cytotoxicity and cytokine production on recognition of self-molecules induced by cellular stress under different conditions such as viral infections. The importance of NKG2D in the immune response to human cytomegalovirus (HCMV) is supported by the identification of several viral molecules that prevent the expression of NKG2D ligands by infected cells. In this study we report that,paradoxically,a significant,selective,and transient reduction of NKG2D expression on NK cells is detected during HCMV infection of peripheral blood mononuclear cells if needed. Antagonizing type I interferon (IFN),interleukin-12 (IL-12),and IFNgamma prevented HCMV-induced down-regulation of surface NKG2D. Moreover,treatment of purified NK cells with recombinant IFNbeta1 and IL-12 mimicked the effect,supporting a direct role of these cytokines in regulating NKG2D surface expression in NK cells. The loss of NKG2D expression selectively impaired NK-cell cytotoxicity against cells expressing NKG2D ligands but preserved the response triggered through other activating receptors. These results support that down-regulation of NKG2D expression on NK cells by cytokines with a key role in antiviral immune response may constitute a physiologic mechanism to control NK-cell reactivity against normal cells expressing NKG2D ligands in the context of inflammatory responses to viral infections. View Publication

The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-lambda1 (interleukin-29 [IL-29]),IFN-lambda2 (IL-28A),and IFN-lambda3 (IL-28B),each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-lambda producers among peripheral lymphocytes. Recently,it has been shown that IFN-lambda1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4(+) T cells and inhibiting the production of Th2-associated cytokines. Here,we asked whether Th2 cytokines exert reciprocal control over IFN-lambda production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-lambda1 transcription and secretion. However,pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments,we showed that IL-4-responsive monocytes are an intermediary cell,responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-lambda1 output. Thus,our experiments revealed a novel mechanism for regulation of both IFN-lambda1 production and pDC function,and suggests an expanded immunomodulatory role for Th2-associated cytokines. View Publication

BACKGROUND AIMS Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products,which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities,there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment,and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS This study compared cryopreservation of PBSC in a novel intracellular-like,fully defined,serum- and protein-free preservation solution,CryoStor (BioLife Solutions Inc.),with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly,human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard,Normosol-R,5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed,and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC),viability,CD34 and granulocytes by flow cytometry,along with colony-forming activity in methylcellulose. RESULTS The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+),colony-forming units (CFU) and granulocytes. Specifically,relative to the FHCRC standard formulation,cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005),a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition,it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins,and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however,these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections,along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO. View Publication

Here,we describe a procedure and first-in-man clinical effects of adoptive transfer of ex vivo expanded CD4+CD25+CD127- T regulatory cells (Tregs) in the treatment of graft versus host disease (GvHD). The cells were sorted from buffy coats taken from two family donors,expanded ex vivo and transferred to respective recipients who suffered from either acute or chronic GvHD. The therapy allowed for significant alleviation of the symptoms and reduction of pharmacologic immunosuppression in the case of chronic GvHD,while in the case of grade IV acute GvHD it only transiently improved the condition,for the longest time within all immunosuppressants used nonetheless. View Publication

IFN-beta-1a has been used over the past 15 years as a primary therapy for relapsing-remitting multiple sclerosis (MS). However,the immunomodulatory mechanisms that provide a therapeutic effect against this CNS inflammatory disease are not yet completely elucidated. The effect of IFN-beta-1a on Th17 cells,which play a critical role in the development of the autoimmune response,has not been extensively studied in humans. We have investigated the effect of IFN-beta-1a on dendritic cells (DCs) and naive CD4(+)CD45RA(+) T cells derived from untreated MS patients and healthy controls in the context of Th17 cell differentiation. We report that IFN-beta-1a treatment down-regulated the expression of IL-1beta and IL-23p19 in DCs,whereas it induced the gene expression of IL-12p35 and IL-27p28. We propose that IFN-beta-1a-mediated up-regulation of the suppressor of cytokine signaling 3 expression,induced via STAT3 phosphorylation,mediates IL-1beta and IL-23 down-regulation,while IFN-beta-1a-induced STAT1 phosphorylation induces IL-27p28 expression. CD4(+)CD45RA(+) naive T cells cocultured with supernatants from IFN-beta-1a-treated DCs exhibited decreased gene expression of the Th17 cell markers retinoic acid-related orphan nuclear hormone receptor c (RORc),IL-17A,and IL-23R. A direct IFN-beta-1a treatment of CD45RA(+) T cells cultured in Th17-polarizing conditions also down-regulated RORc,IL-17A,and IL-23R,but up-regulated IL-10 gene expression. Studies of the mechanisms involved in the Th17 cell differentiation suggest that IFN-beta-1a inhibits IL-17 and induces IL-10 secretion via activated STAT1 and STAT3,respectively. IFN-beta's suppression of Th17 cell differentiation may represent its most relevant mechanism of selective suppression of the autoimmune response in MS. View Publication

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-gamma secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family,such as NKp46,by ligands expressed on tumour cells. However,it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here,we investigated the early events occurring during T cell-tumour cell interactions,and their impact on NK cell functions. We observed that on co-culture with some melanomas,activated CD4(+) T cells promoted degranulation,and NKG2D- and NKp46-dependent IFN-gamma secretion by NK cells,probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4(+),CD8(+) and resting T cells,which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new,so far,unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity. View Publication